Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention discloses the combined treatment of memantine
(N-methyl-D-aspartate receptor antagonist) and tea polyphenol (an
antioxidant and anti-inflammatory agent) is more effective (synergistic)
in neuroprotection than either memantine or tea polyphenol alone in mouse
excitotoxic injury. These findings provide useful information about the
potential application of memantine and tea polyphenols in preventing or
treating clinical excitotoxic injury such as brain trauma, brain
ischemia, epilepsy, and Alzheimer's disease.

Claims:

1. A composition, comprising N-methyl-D-aspartate (NMDA) receptor
antagonist and tea polyphenol in combination, wherein the ratio of tea
polyphenol and NMDA receptor antagonist is from 3:1 to 10:1.

2. The composition according to claim 1, wherein said NMDA receptor
antagonist is memantine or a pharmaceutically acceptable analog.

3. The composition according to claim 1, wherein said tea polyphenol
consists of polyphenolic antioxidant metabolites.

4. A method for preventing or treating a subject suffering from a disease
or condition associated with excitotoxicity, said method comprising
administering to the subject a therapeutically effective amount of the
composition of claim 1.

5. The method according to claim 4, wherein said preventing or treating
is made by attenuating mitochondria dysfunction associated with loss of
Ca2+ homeostasis and enhanced cellular oxidative stress.

6. The method according to claim 4, wherein said disease or condition is
a neurodegenerative disease or condition.

7. The method according to claim 6, wherein said neurodegenerative
disease or condition is brain trauma, brain ischemia, epilepsy, or
Alzheimer's diseases.

8. The method according to claim 4, wherein said excitotoxicity is caused
by NMDA receptor over-activation.

9. The method according to claim 4, wherein said subject is human.

10. A method for providing synergistic neuroprotective effect to a
subject, said method comprising administrating to the subject a
therapeutically effective amount of the composition of claim 1.

11. The method according to claim 12, wherein said synergistic
neuroprotective effect is made by attenuating mitochondria dysfunction
associated with loss of Ca2+ homeostasis and enhanced cellular
oxidative stress.

12. The method according to claim 12, wherein said subject is human.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation-in-part of the pending U.S.
patent application Ser. No. 12/243,655 filed on Oct. 1, 2008, for which
priority is claimed and is incorporated herein by reference in its
entirety.

[0002] Although incorporated by reference in its entirety, no arguments or
disclaimers made in the parent application apply to this divisional
application. Any disclaimer that may have occurred during the prosecution
of the above-referenced application(s) is hereby expressly rescinded.
Consequently, the Patent Office is asked to review the new set of claims
in view of the entire prior art of record and any search that the Office
deems appropriate.

FIELD OF THE INVENTION

[0003] The present invention relates to a composition and a method for
synergistic neuroprotection against excitotoxic injury.

BACKGROUND OF THE INVENTION

[0004] Glutamate is the main excitatory neurotransmitter in the mammalian
central nervous system (CNS) and mediates neurotransmission in most
excitatory synapses. Three classes of glutamate-gated ion channel
receptors--α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA), kainite, and N-methyl-D-aspartate (NMDA) receptors--can transduce
postsynaptic signals. Among them, NMDA receptors are the most abundant
and ubiquitously distributed throughout the brain. Therefore, they are
fundamental to excitatory neurotransmission and critical for the
maintenance of normal CNS function. However, excessive glutamate
overstimulates NMDA receptors, leading to increased intracellular calcium
and excitotoxicity (Kemp J A, McKernan R M. 2002. NMDA receptor pathways
as drug targets. Nat Neurosci 5: 1039-1042.). It is by the
glutamate-dependent mechanism that neurons die in various CNS disorders,
including brain ischemia, epilepsy, and Alzheimer's disease. The role of
NMDA receptors in excitotoxicity has driven the search for antagonists as
neuroprotective agents.

[0005] On the other hand, NMDA receptor activity is essential for normal
neuronal function. Potential neuroprotective agents that block virtually
all NMDA receptor activity lead to unacceptable clinical side effects
(drowsiness, hallucination, and even coma) because they block normal NMDA
receptor activity (Palmer G C. 2001. Neuroprotection by NMDA receptor
antagonists in a variety of neuropathologies. Curr Drug Targets 2:
241-271.). For this reason, many previous NMDA receptor antagonists have
failed advanced clinical trials in a number of neurodegenerative
disorders. In contrast, some studies have shown that the adamantane
derivative memantine can block excessive NMDA receptor activity without
disrupting normal activity and shows promise in clinical applications
(Chen H S, Lipton S A. 2006. The chemical biology of clinically tolerated
NMDA receptor antagonists. J Neurochem 97: 1611-1626.). Memantine exerts
its pharmacological effects through its action as a low-affinity,
uncompetitive open-channel blocker. Memantine has unique blocking sites
in channel pores, and this subtle difference between memantine and other
traditional NMDA receptor antagonists may explain many advantageous
properties of memantine action. In fact, in normal conditions, the
excitatory postsynaptic current resulting from physiological activation
of NMDA receptors is mostly preserved. In excitotoxic conditions, when
prolonged activation of NMDA receptors occurs, memantine becomes a very
effective blocker. In essence, the pharmacological effects of memantine
are most obvious under pathological conditions, and it maintains the
normal functions of receptors, thus relatively sparing synaptic
transmission and preserving long-term potentiation and maintaining
physiological function (Chen H S, Wang Y F, Rayudu P V, Edgecomb P, Neill
J C, Segal M M, Lipton S A, Jensen F E. 1998a. Neuroprotective
concentrations of the N-methyl-D-aspartate open-channel blocker memantine
are effective without cytoplasmic vacuolation following post-ischemic
administration and do not block maze learning or long-term potentiation.
Neuroscience 86: 1121-1132.). In fact, memantine has been used clinically
with an excellent safety record for more than 20 years in Europe to treat
Parkinson's disease, spasticity, convulsions, vascular dementia, and
Alzheimer's disease.

[0020] The present invention discloses a composition comprising
N-methyl-D-aspartate (NMDA) receptor antagonist and tea polyphenol in
combination, wherein the ratio of tea polyphenol and NMDA receptor
antagonist is from 3:1 to 10:1.

[0021] The present invention also discloses a method for preventing or
treating a subject suffering from a disease or condition associated with
excitotoxicity resulted from NMDA receptor over-activation, said method
comprising administering to the subject a therapeutically effective
amount of the composition of the present invention.

[0022] The present invention further discloses a method for providing
synergistic neuroprotective effect to a subject, said method comprising
administrating to the subject a therapeutically effective amount of the
composition of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This invention demonstrated that intrastriatal injection of NMDA
caused impairment in locomotor activity, increased production of
synaptosomal ROS, and a decrease in all Na+, K+-ATPase and
Mg2+-ATPase activity, mitochondrial membrane potential
(ΔΨm), and mitochondrial reductase activity in mice. Treatment
with tea polyphenol could significantly decrease the increased
synaptosomal ROS production, and attenuate the decreased Na+,
K+-ATPase and Mg2+-ATPase activity. By contrast, treatment with
memantine could significantly attenuate the decreased mitochondrial
membrane potential (ΔΨm) and mitochondrial reductase activity.
However, neither memantine nor tea polyphenol alone could significantly
improve the impaired locomotor activity after excitotoxic injury. A
promising regimen through combination of memantine and tea polyphenol
significantly improved locomotor activity, decreased synaptosomal ROS
production, and attenuated all the decreases in Na+, K+-ATPase
and Mg2+-ATPase activity, mitochondrial membrane potential
(ΔΨm), and mitochondrial reductase activity in mouse
excitotoxic injury. Therefore, it is suggested that memantine, an NMDA
receptor antagonist, combined with tea polyphenol, an antioxidant, is
more effective in neuroprotection than is either alone in mouse
excitotoxic injury.

[0024] Excitotoxicity is an attractive target for neuroprotective efforts
because it is involved in the pathophysiology of a wide variety of acute
and chronic neurological disorders. Trying to devise strategies for
combating excitotoxicity is still a challenging task because the same
processes that in excess lead to excitotoxic cell death are absolutely
critical for normal neuronal function at lower levels. To be clinically
acceptable, an NMDA receptor antagonist must block excessive activation
of NMDA receptors while leaving normal function relatively intact in
order to avoid side effects. The term open-channel blocker of the NMDA
receptor means that the drug enters the receptor-associated ion channel
only when it is open. Importantly, this type of drug will be most
effective in the face of excessive (pathological) activity. This
mechanism of inhibition, whose action depends on prior activation of the
receptor by the agonist, is defined as uncompetitive antagonism. For
therapeutic intervention during excessive NMDA receptor activation,
open-channel block is a very appealing strategy because the action of the
blockade requires prior activation of the receptors. This property can
lead to a higher degree of channel blockade in the presence of excessive
levels of glutamate and only little blockade at relatively lower levels
to maintain physiological neurotransmission (Chen H S, Lipton S A. 2006.
The chemical biology of clinically tolerated NMDA receptor antagonists. J
Neurochem 97: 1611-1626.). Memantine is an ideal NMDA receptor antagonist
through its action as a low-affinity, uncompetitive open-channel blocker.

[0025] In the present invention, treatment with memantine could
significantly attenuate the decreased mitochondrial membrane potential
(ΔΨm) and mitochondrial reductase activity in mouse excitotoxic
injury. Memantine does not have any significant effects on increased
synaptosomal ROS production and decreased Na+, K+-ATPase and
Mg2+-ATPase activity. Therefore, treatment with memantine alone
caused nonsignificant improvement in the impaired locomotor activity.
Energy depletion is among the frequent initiating conditions leading to
excitotoxicity, and mitochondrial dysfunction is believed to be one of
the most generalized causes favoring the development of neurodegenerative
diseases. Memantine has been tested in animals against primary insults
dependent on mitochondrial impairment and energy depletion and has
provided protection from inhibition of mitochondrial function (Rego A C,
Oliveira C R. 2003. Mitochondrial dysfunction and reactive oxygen species
in excitotoxicity and apoptosis: implications for the pathogenesis of
neurodegenerative diseases. Neurochem Res 28: 1563-1574.). Therefore, the
neuroprotective effect of memantine in excitotoxic injury depends on
amelioration of mitochondrial dysfunction, which was well demonstrated in
our experiment. However, memantine lacks the ability to scavenge the
excessive production of ROS and its associated toxic outcomes. In an
excitotoxic injury model with an acutely high concentration of NMDA, as
in our experiment or in acute stroke, memantine alone is not adequate for
significant neuroprotection.

[0026] It is well documented that mitochondrial dysfunction associated
with the loss of Ca2+ homeostasis and enhanced cellular oxidative
stress has long been recognized to play a major role in cell damage
associated with excitotoxicity. Na+, K+-ATPase is known to be
highly susceptible to free-radical damage and lipid peroxidation because
of its plasmalemmal embedding and its phospholipid requirement for
maintenance of activity (Ildan F, Polat S, Gocer A I, Oner A, Isbir T,
Mete U O, Kaya M, Karadayi A. 1996. The effects of the pretreatment of
intravenous high dose methylprednisolone on
Na+--K+/Mg2+-ATPase and lipid peroxidation and early
ultrastructural findings following middle cerebral artery occlusion in
the rat. Acta Neurochir (Wien) 138: 338-345.). Mg2+-ATPase is also
vulnerable to damage by reactive oxygen species. It is therefore proposed
that failure of memantine to attenuate the decrease in Na+,
K+-ATPase and Mg2+-ATPase activity is a result of its inability
to scavenge the increased production of ROS.

[0028] This invention disclosed a novel regimen to use, the combined
treatment of memantine and tea polyphenol, in excitotoxic injury. The
results showed significant protection against excitotoxic injury with
combined treatment with memantine and tea polyphenol. The neuroprotective
effects included reduction in increased synaptosomal ROS and Ca2+
concentration and attenuation of decreased Na+, K+-ATPase and
Mg2+-ATPase activity, mitochondrial membrane potential
(ΔΨm), and mitochondrial reductase activity. Moreover,
impairment in locomotor activity was also significantly improved.
Therefore, combined treatment with memantine and tea polyphenol is more
effective in neuroprotection than memantine or tea polyphenol alone in
mouse excitotoxic injury.

[0029] Combined treatment with memantine and tea polyphenol is more
effective in neuroprotection than memantine or tea polyphenol alone in
mouse excitotoxic injury. Further experiments are needed to explore the
efficacy of such a novel regimen in the treatment of neurodegenerative
and other neurological diseases.

[0030] Accordingly, the present invention discloses a composition,
comprising N-methyl-D-aspartate (NMDA) receptor antagonist and tea
polyphenol in combination and the ratio of tea polyphenol and NMDA
receptor antagonist is from 3:1 to 10:1. In a preferred embodiment of the
composition, NMDA receptor antagonist is memantine or a pharmaceutically
acceptable analog and tea polyphenol consists of polyphenolic antioxidant
metabolites. In another preferred embodiment of the composition, the
ratio of tea polyphenol and NMDA receptor antagonist is 10:1

[0031] The present invention also discloses a method for preventing or
treating a subject suffering from a disease or condition associated with
excitotoxicity resulted from NMDA receptor over-activation, said method
comprising administering to the subject a therapeutically effective
amount of the composition of the present invention, wherein preventing or
treating is made by attenuating mitochondria dysfunction associated with
loss of Ca2+ homeostasis and enhanced cellular oxidative stress. In
a preferred embodiment of the method, the disease or condition associated
with excitotoxicity resulted from NMDA receptor over-activation is
neurodegenerative disease or condition.

[0032] In addition, the neurodegenerative disease or condition is brain
trauma, brain ischemia, epilepsy, or Alzheimer's diseases. And the
subject to be administered is human.

[0033] The present invention further discloses a method for providing
synergistic neuroprotective effect, said method comprising administrating
to the subject a therapeutically effective amount of the composition of
the present invention. The synergistic neuroprotective effect is made by
attenuating mitochondria dysfunction associated with loss of Ca2+
homeostasis and enhanced cellular oxidative stress. And the subject to be
administered is human.

[0034] The term "synergistic neuroprotective effect" is not limited but to
make through attenuating mitochondria dysfunction associated with loss of
Ca2+ homeostasis and enhanced cellular oxidative stress.

EXAMPLES

Example 1

Mice

[0035] The experiment protocols were approved by the Hospital Animal
Research Committee of National Taiwan University Hospital. Adult male ICR
mice weighing 20-25 g were used in this example.

[0037] Weight and rectal temperature of each mouse were recorded before
the surgical procedure. Anesthesia was induced with 5% chloral hydrate
(400 mg/kg). Each mouse was mounted on a stereotactic frame, and 0.3 L of
NMDA (335 mM, pH, 7.2) prepared in phosphate-buffered saline was injected
into the left striatum (stereotactic coordinates: PA 0.5 mm, lateral 3.0
mm from bregma, and ventral 4 mm relative to dura) over a 2-mM period;
the needle was left in situ for an additional 5 mM to prevent backflow.
All five groups of mice received the same procedure except that the same
amount of normal saline was injected in the sham operation group. After
injections, mice were placed in a humidified, thermoregulated chamber
maintained at 31° C. and were returned to their cages after full
recovery from anesthesia. Throughout the experimental procedure, mouse
rectal temperature was monitored and maintained at 37.0°
C.±0.5° C.

Preparation of Synaptosomes

[0038] Twenty-four hours after excitotoxic injury, mice were sacrificed by
rapid decapitation under anesthesia. The lesioned (left) hemisphere was
dissected into striatal and nonstriatal areas. The contralateral
hemisphere was then dissected into the corresponding parts. Synaptosomes
were prepared essentially as described previously (Andersen J M, Myhre O,
Formum F. 2003. Discussion of the role of the extracellular
signal-regulated kinase-phospholipase A2 pathway in production of
reactive oxygen species in Alzheimer's disease. Neurochem Res 28:
319-326.). Briefly, different regions of the brain were removed and
placed on ice. Specimens with the same areas and treatment conditions
were pooled (n=2) and subjected to homogenization on ice in 10 volumes of
0.32M sucrose. Homogenates were centrifuged at 4° C. for 10 min at
600 g. The supernatant was then diluted 1:1 with 1.3M sucrose to obtain a
suspension with a final sucrose concentration of 0.8M. This suspension
was subjected to centrifugation at 20,000 g for 30 min at 4° C.,
yielding a myelin-rich supernatant and a pellet (P2) consisting of
synaptosomes free of myelin. The supernatant was discarded, and the
pellet was resuspended in 0.32M sucrose buffer (pH 7.4). Synaptosomes
were held on ice, usually for 15-20 min, until experiments were
performed. The concentration of synaptosomes used for the experiments was
corrected as milligrams of protein.

Statistical Analysis

[0039] Statistical comparisons between study groups were performed using
analysis of variance followed by Dunnett's test. P values less than 0.05
were considered biologically significant.

Locomotor Activity Test

[0040] All experiment animals received a locomotor activity test 1 day
before and 1 day after excitotoxic injury.

Result:

[0041] Locomotor Activity was Impaired after Excitotoxic Injury and
Attenuated by Combined Treatment with Memantine and Tea Polyphenol.

[0042] The administration of NMDA in mouse striatum caused significant
impairment of locomotor activity. Locomotor activity tests administered
to the experimental mice were recorded 24 hr before and after excitotoxic
injury. In the control group, a significant decrease was noted in
ambulation distance, jump, and vertical plane entry after excitotoxic
injury (n=8, P<0.01; FIG. 1). An increase in rest time was also noted.
Treatment with memantine or tea polyphenol alone caused substantial but
nonsignificant improvement in the impaired locomotor activity. Combined
treatment with memantine and tea polyphenol could significantly protect
mice from impairment of locomotor activity after excitotoxic injury (n=8,
P<0.05; FIG. 1).

Measurements of Na+, K+-ATPase Activity

[0043] ATPase activity was determined by measuring the amount of inorganic
phosphate (Pi) released from the substrate ATP according to a previously
described colorimetric method. The method permitted the quantification of
Na+, K+-ATPase and Mg2+-ATPase activity in the same
sample. Briefly, ATPase reactions were initiated in a mixture containing
NaCl (354 mM), KCl (14.4 mM), MgCl2 (3.6 mM), NaHCO3 (37.5 mM),
ethyleneglycol bis(amino-ethylether) tetraacetate (1.5 mM), glucose (33.3
mM), and ATP (9 mM) and in the absence or presence of ouabain (1 mM).
Synaptosomes prepared from different areas (striatum and nonstriatum) of
the same brain were incubated at 37° C.±0.5° C. for 30
min in the reaction mixture. Reactions were terminated by the addition of
150 L of a solution containing ammonium molybdate (1.05%), malachite
green hydrochloride (0.034%), and Triton-X (0.6%). To stabilize the color
reaction, 10 L of a sodium citrate solution (34%) was added, and the
assay solution was held at room temperature for 20 min Optical density at
630 nm was determined by an ELISA reader (Dynatech MR-7000). The
absorbance values obtained were converted to activity values by linear
regression using a standard curve for sodium monobasic phosphate included
in the assay at various concentrations. Pi released (in mmol/L) was taken
to represent the concentration of inorganic phosphate released by the
enzymatic hydrolysis of ATP. Na+, K+-specific ATPase activity
was determined by subtracting ouabain-insensitive Mg2+-ATPase
activity from total Na+, K+ and Mg2+-ATPase activity
(Cheng P W, Liu S H, Hsu C J, Lin-Shiau S Y. 2005. Correlation of
increased activities of Na+, K+-ATPase and Ca2+-ATPase
with the reversal of cisplatin ototoxicity induced by D-methionine in
guinea pigs. Hear Res 205: 102-109.). Protein concentration was
determined colorimetrically with a commercial bicinchoninic acid kit
(Pierce, Rockford, Ill.).

Result:

[0044] Na+, K+-ATPase and Mg2+-ATPase Activity Decreased
after Excitotoxic Injury and Was Preserved by Treatment with Tea
Polyphenol or Combined Treatment of Memantine and Tea Polyphenol.

[0046] Dichlorofluorescein-diacetate (DCFH-DA), a nonfluorescent
cell-permeable compound, diffuses passively across cell membranes.
Following cellular uptake, the acetate groups are cleaved by
intracellular esterases, yielding 2,7-dichlorofluorescin (DCFH). DCFH is
oxidized by hydroxyl radicals, peroxynitrite, or H2O2 (in the
presence of peroxidases) to a fluorescent compound,
2,7-dichlorofluorescein (DCF; Myhre O, Andersen J M, Aarnes H, Formum F.
2003. Evaluation of the probes 2,7-dichlorofluorescin diacetate, luminol,
and lucigenin as indicators of reactive species formation. Biochem
Pharmacol 65: 1575-1582.). Production of the latter was therefore used as
an index of reactive oxygen species formation. Synaptosomes were diluted
1:40 in 0.32M sucrose buffer prior to loading with 10 M DCFH-DA for 15
min at 37° C. Working solutions were prepared daily by diluting
stock solutions in 0.32M sucrose buffer to 1.67× the desired final
concentration; 150 L of this working solution was then placed in wells of
96-well microplates. The reaction was initiated by the addition of 100 L
of the synaptosomal solution to each well (final reaction volume of 250
L). Plates were incubated with shaking at 37° C. for 30 min before
measurement of fluorescence using a Microplate Fluorometer (Labsystems,
Helsinki, Finland) with an excitation wavelength of 488 nm, an emission
wavelength of 525 nm, and a band width of 5 nm Blank values were obtained
from wells containing buffer and synaptosomes that had not been loaded
with DCFH-DA. Synaptosomal protein concentration was determined with
Pierce BCA reagents according to instructions provided. DCF fluorescence
values were corrected for protein values and autofluorescence of the
samples according to the formula Fco=(Fsa-Fbl)/synaptosomal protein,
where Fco was the corrected fluorescence value, Fsa was the observed
fluorescence of the sample, and Fbl was the observed fluorescence of the
blank.

Result:

[0047] Increased Production of Reactive Oxygen Species after Excitotoxic
Injury and Decreased Production by Treatment with Tea Polyphenol or
Combined Treatment with Memantine and Tea

Polyphenol

[0048] In striatum, the synaptosomal production of reactive oxygen species
was significantly increased after excitotoxic injury (n=8, P<0.05).
Similar to ATPase, treatment with tea polyphenol alone or combined
treatment with memantine and tea polyphenol significantly decreased the
production of synaptosomal reactive oxygen species (treatment with tea
polyphenol and combined treatment with memantine and tea polyphenol vs.
control group: 74.57%±7.72% and 70.65%±7.97% vs. 92.83%±6.65%,
P<0.05; FIG. 3).

Mitochondrial Membrane Potential

[0049] Mitochondrial membrane potential (ΔΨm) was measured using
the fluorescent dye 3,3-diexyloxacarbocyanine iodide, DiOC6(3) (Chen Y C,
Lin-Shiau S Y, Lin J K, 1998b. Involvement of reactive oxygen species and
caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 177:
324-333.). Synaptosomes were prepared as described above and diluted 1:40
in Tris buffer, followed by the addition of DiOC6(3) to a final
concentration of 1.5 M. After 20 min of incubation at 37° C.,
fluorescence was measured in a Microplate Fluorometer (Labsystems,
Helsinki, Finland) with an excitation wavelength of 484 nm and an
emission wavelength of 501 nm.

Result:

[0050] Mitochondrial Membrane Potential (ΔΨm) Decreased after
Excitotoxic Injury and Was Preserved by Treatment with Memantine or
Combined Treatment with Memantine and Tea Polyphenol

[0052] Mitochondrial metabolic function was assessed by the conversion of
the dye methylthiazoletetrazolium (MTT) to purple formazan. This assay
was based on the ability of mitochondrial succinate reductase to
metabolize MTT to formazan; this reaction took place only in functionally
intact mitochondria. P2 synaptosomes were prepared as described above.
Working solutions (150 L) were added to microtubes, and 100 L of the
synaptosome suspension and 25 L of a 5.0 mg/mL solution of MTT in 0.32M
sucrose buffer were added to each tube. The samples were incubated for
120 min at 37° C. The purple formazan crystals were pelletized by
centrifugation and the supernatant discarded. The pellets were dissolved
in dimethyl sulfoxide and transferred to 96-well microplates. The
formation of formazan was quantitated spectrophotometrically at 570 nm
using a Microplate Reader.

Result:

[0053] Mitochondrial Reductase Activity Decreased after Excitotoxic Injury
and Was Attenuated by Treatment with Memantine or Combined Treatment with
Memantine and Tea Polyphenol. Mitochondrial reductase activity (MTT test)
was measured 24 hr after excitotoxic injury. In the control group, the
reductase activity decreased significantly after excitotoxic injury (n=8,
P<0.01). Treatment with memantine or combined treatment with memantine
and tea polyphenol significantly attenuated the decrease in reductase
activity (treatment with memantine and combined treatment with memantine
and tea polyphenol vs. control group: 93.53%+8.53% and 96.65%+6.42% vs.
74.52%±4.86%, P<0.05; FIG. 5).

Measurement of Intrasynaptosomal Ca2+ Concentration

[0054] Intrasynaptosomal Ca2+ concentration [Ca2+]i was
determined with the calcium-sensitive fluorochrome fluo-3-acetoxymethyl
(Fluo-3/AM) in the presence of acetoxymethyl ester. First, 100 L of P2
synaptosomes was prepared as described above, and 2.5 mM Fluo-3/AM
(Sigma, St. Louis, Mo.) was added to each well (final reaction volume of
250 L) and incubated at 37° C. for 30 min. Fluorescence was
measured in a Microplate Fluorometer (Labsystems, Helsinki, Finland) with
an excitation wavelength of 490 nm and an emission wavelength of 526 nm

Result:

[0055] Intrasynaptosomal Ca2+ Concentration Increased after
Excitotoxic Injury and Was Attenuated by Treatment with Memantine and Tea
Polyphenol Alone or in Combination.

[0057] The male mice (ICR strain) weighting 18-20 g were housed at
22±1° C., under a 12 h light-dark cycle with food and water
available ad libitum. Animals were habituated to the housing conditions
for one week prior to the experiments. Behavioral testing was carried out
during the light portion of the cycle. The experimental protocols were
approved by the Hospital Animal Research Committee of National Taiwan
University Hospital.

Isolation and Preparation of Tea Polyphenols

[0058] TP (tea polyphenol) was isolated according to the procedure
described in example 1.

Statistic

[0059] Statistical comparisons between study groups were performed using
one-way ANOVA test followed by post hoc multiple comparison with
Dunnett's test. Factors are different groups. In comparing seizure
frequency, Chi square tests were performed. P values of less than 0.05
were considered to be statistically significant.

[0062] In this example, the combination of TP with M (memantine) in three
different dosage ratios of 6:1, 3:1, and 10:1 respectively; these are
TPM6 (TP 30 mg/kg/day:M 5 mg/kg/day), TPM3 (TP 15 mg/kg/day:M 5
mg/kg/day) and TPM10 (TP 30 mg/kg/day:M 3 mg/kg/day) have been designed.
Their neuroprotective effects were evaluated after pretreatment for
fourteen days and one day prior to the intracerebral injection (icy) of 8
mM NMDA (3 μl). The results showed that attenuations of NMDA-induced
seizure scores in percentage by M, TPM6, TPM3 and TPM10 were by
60±10%, 50±12%, 60±13% and 70±10% respectively, as compared
with the control vehicle 90±10%. It appeared that the drugs treatments
were all effectively in decreasing NMDA-induced excitotoxic seizures and
TPM10 apparently was superior to M alone or TPM6 or TPM3.

Locomotor Activity Test

[0063] Mice were individually placed in an open field and performed in a
separated room with no interference noise or human activity as described
previously (Chuu et al. 2001. Abnormal auditory brainstem responses for
mice treated with mercurial compounds: involvement of excessive nitric
oxide. Toxicology 162: 11-22.). A large colorless rectangular box with a
metallic grid floor was used (70-cm wide, 90-cm long and 60-cm high). The
photobeam activity monitors (TruScan coulbourn instruments) was used for
measuring real-time X-Y activity track-type plots. Overall pulses were
measured in an electromechanical counter as a gross measure of activity
and recorded by a PC. Each mouse was allowed to move freely for 5 min but
data were not scored, and then the number of squares crossed and the
plots of tracking were counted during a period of 30 min for all
experiments and quantification of data was by TruScan99 software.

Elevated Plus Maze

[0064] For testing, mice were individually placed in the center of the
maze facing a closed arm and allowed 5 min of free exploration. The
number of entries into open arms, the number of entries into closed arms,
and the total time spent in the open arms and total time spent in the
closed arms were measured. Entry was defined as all four paws in the
arms.

Rotarod Motor Equilibrium Performance on Rotarod

[0065] Mice were tested for their ability to balance on a slowly rotating
rod (60 revolutions per min) as described previously (Chuu et al. 2001.
Abnormal auditory brainstem responses for mice treated with mercurial
compounds: involvement of excessive nitric oxide. Toxicology 162:
11-22.). One day before the experiment, mice were tested through ten
consecutive sessions to stay on the rod and reach the cut off time of 180
s. One day after injection, mice were tested again. The retention time,
defined as total time (sec) remaining on the rod, was recorded at each
session.

Results:

Effects on Locomotor Activities

[0066] As shown in FIG. 7, memantine (10 mg/kg/day) for consecutive 14
days increased locomotor activities in the open field, TPM6 and TPM3 only
slightly increased and TPM10 had no effect on the locomator activities.
After icy injection of 3 μl of 8 mM NMDA markedly decreased locomotor
activities which were prevented by pretreatment with M or TPM. Estimation
of the neuroprotective potencies against NMDA-decreased locomotor
activities (FIG. 8A), the orders of the potencies against NMDA
neurotoxicities were TPM10>TPM6>TPM3>M. As to the
neuroprotective effects against the decreased jump exploratory effect of
NMDA (FIG. 8B), the order of the potencies are
M>TPM6>TPM3>TPM10. However, after pretreatment with M and TPM
alone for consecutive 14 days, the decrease in jump activity in orders
are TPM6>M>TPM10 and TPM3 did not affect the jump activities (FIG.
8B). In addition, NMDA increased the retention time in the open field of
elevated plus maze (FIG. 8C) but decreased rotarod motor equilibrium
function which could be most efficiently prevented by TPM10. It was noted
that prolonged use of M alone prominently increased retention time in the
open field of elevated plus maze and reduced (FIG. 8C) the motor
equilibrium function similarly to NMDA (FIG. 8C). Both TPM6 and TPM3 only
slightly inhibited NMDA in open field and completely prevented
NMDA-induced disturbance in motor equilibrium function (FIG. 8D). On the
other hand, treatment of the drugs for one day and then icy injection of
NMDA, the induced excitotoxic effects were persistent and the
neuroprotective effects of the drugs were investigated by administration
for consecutive 14 days following NMDA administration (FIG. 9). The
comparative neuroprotective effects of the different dosage regimens were
shown in FIG. 10A-10D. The decreased locomotor activities induced by NMDA
could be partially reversed by M and TPM6, while TPM3 and TPM10 appeared
to completely reverse and then further increasing excitatory achieving to
120% of the control (FIG. 10A). The decrease in jump activity induced by
NMDA could be partially reversed by M and TPM6, while TPM3 reversed over
to 160% and TPM10 exhibited the optimal neuroprotection achieving to the
control level (FIG. 10B). Similarly, the increased retention time in the
opened plus maze by NMDA was attenuated the best by TPM3, followed in
order by TPM6, TPM10 and then M (FIG. 10C). The disturbance in rotarod
motor equilibrium function induced by NMDA could be reversed by M and
TPM10 but not by TPM6 and TPM3 (FIG. 10D).

Biochemical Analysis of Brain Tissues

[0067] a. Preparation of Synaptosomes

[0068] The mice were sacrificed by rapid decapitation under anesthesia.
The brain was dissected into four parts: cerebral cortex (CC), striatum
(St), cerebellar cortex (CB), and brain stem (BS). Synaptosomes were
prepared essentially as described in example 1. Briefly, different
regions of the brain were removed and placed on ice. Specimens with the
same areas and treatment conditions were subjected to homogenization on
ice in 10 volumes of 0.32 M sucrose. Homogenates were centrifuged at
4° C. for 10 min at 600×g. The supernatant was then diluted
1:1 with 1.3 M sucrose to obtain a suspension with a final sucrose
concentration of 0.8 M. This suspension was subjected to centrifugation
at 20,000 g for 30 min at 4° C., yielding a myelin-rich
supernatant and a pellet consisting of synaptosomes free of myelin. The
supernatant was discarded, and the pellet was resuspended in 0.32 M
sucrose buffer (pH 7.4). Synaptosomes were held on ice, usually for 15-20
min, until experiments were performed. The concentration of synaptosomes
used for the experiments was corrected as mg proteins. The
neurobiological and behavioral measures were taken on the same mice.

b. Measurements of Na+, K+-ATPase Activity

[0069] ATPase activities were determined by measuring the amount of
inorganic phosphate (Pi) released from the substrate ATP according to a
previously described colorimetric method. The method permitted the
quantification of Na+, K+-ATPase and Mg2+-ATPase
activities in the same sample. Briefly, ATPase reactions were initiated
in a mixture containing NaCl (354 mM), KCl (14.4 mM), MgCl2 (3.6 mM),
NaHCO3 (37.5 mM), ethyleneglycol bis(amino-ethylether) tetraacetate
(EGTA, 1.5 mM), glucose (33.3 mM) and ATP (9 mM), and in the absence or
presence of ouabain (1 mM). Synaptosomes prepared from different brain
areas were incubated at 37±0.5° C. for 30 min in the reaction
mixture. Reactions were terminated by the addition of 150 μl of a
solution containing ammonium molybdate (1.05%), malachite green
hydrochloride (0.034%) and Triton-X (0.6%). To stabilize the color
reaction, 10 μl of a sodium citrate solution (34%) was added, and the
assay solution was held at room temperature for 20 mM The optical density
at 630 nm was determined by an ELISA reader (Dynatech MR-7000). The
absorbance values obtained were converted to activity values by linear
regression using a standard curve for sodium monobasic phosphate included
in the assay at various concentrations. Pi released (in mmol/l) was taken
to represent the concentration of inorganic phosphate released by the
enzymatic hydrolysis of ATP. Na+, K+-specific ATPase activity
was determined by subtracting ouabain-insensitive Mg2+-ATPase
activity from total Na+, K+ and Mg2+-ATPase activities.
Protein concentration was determined colorimetrically with a commercial
bicinchoninic acid kit (Pierce, Rockford, Ill.).

c. Mitochondrial Membrane Potential

[0070] Mitochondrial membrane potential (ΔΨm) was measured using
the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)].
Synaptosomes were prepared as described above and diluted 1:40 in sucrose
buffer, followed by addition of DiOC(6) to a final concentration of 1.5
μM. After 20 min of incubation at 37° C., fluorescence was
measured in a Microplate Fluorometer (Labsystems, Helsinki, Finland)
(excitation wavelength: 484 nm; emission wavelength: 501 nm).

d. Assessment of Mitochondrial Metabolic Function

[0071] Mitochondrial metabolic function was assessed by the conversion of
the dye methylthiazoletetrazolium (MTT) to purple formazan. This assay is
based on the ability of mitochondrial succinate reductase to metabolize
MTT to formazan; this reaction takes place only in functionally intact
mitochondria. Synaptosomes were prepared as described above. 100 μl of
the synaptosome suspension and 50 μl of 5.0 mg/ml solution of MTT in
0.32 M sucrose buffer were added to each tube. The samples were incubated
for 120 min at 37° C. The purple formazan crystals were pelleted
by centrifugation and the supernatant discarded. The pellets were
dissolved in dimethylsulfoxide and transferred to 96-well microplates.
The formation of formazan was quantitated spectrophotometrically at 570
nm using a Microplate Reader.

e. Nitric Oxide Detection

[0072] The brainstems used herein were weighed and homogenized in 10%
(w/v) of homogenate buffer (10% sucrose buffer), and then centrifuged at
0° C. for 20 min at 10000×g. To avoid incomplete protein
denaturation, we added 70% ethanol into the tissue pellet and also the
blood sample, and allowed the mix to stand overnight. On the following
day, all samples were centrifuged at 4±0° C. for 2 min at
12000×g, the supernatants from the brainstem tissue and the whole
blood being collected and assayed by a NO/ozone chemiluminescence assay
method (NO-Analyzer 280A, Sievers Research Inc., Boulder, Colo., USA) for
quantitative NO assay. Briefly, we measured the oxidation products
nitrite (NO*2) of NO using a reaction vessel containing a reducing system
(0.1 M vanadium chloride, Aldrich Co., Germany). The detection of NO is
completed by its reaction with ozone, which leads to the emission of red
light (NO+O3NO*2+O2; NO*2→ NO2+hν). The
linearity of the standard curve was confirmed with 1, 5, 10, 15 and 20
μM NO, these being prepared using freshly-prepared solutions of
NaNO2 (10 μl) in distilled water. The brainstems of the Hg
treated mice were acquired and homogenized for the purposes of the
determination of NOχ (NO.sup.-2 plus.sup.-3) levels
immediately, 5 and 11 weeks subsequent to the cessation of Hg
administration.

Results

[0073] As shown in FIGS. 11A and 11B, NMDA decreased mitochondrial
reductase (MTT) and nitroblue tetrazolium (NBT) activities, which could
be better reversed by administration of TPM 10 and TPM6 followed by M and
TPM3. The mitochondrial membrane potential was slightly decreased by NMDA
which was elevated by TPM10 and M to the normal control level but
unaltered by TPM6 and TPM3 (FIG. 11C). The decrease in Na+--K+
ATPase activities (the most sensitive biomarker to reactive oxygen
species) by NMDA could be reversed by TPM3 and TPM10 followed by TPM6 but
not by M (FIG. 11D). The NO levels of brain synaptosomes were increased
by NMDA which was also reversed by TPM3 and TPM10 but decreased by M and
TPM6 (FIG. 11E).

[0074] Memantine is currently recognized as a useful clinical drug for
improving cognition function of Alzheimer disease patients. However, the
side effects of long term administration of memantine such as
hallulination, delusion and psychosis should be awared for combating.
Examples 1 and 2 confirmed the fact that tea polyphenol could not only be
neuroprotective but also potentiate memantine against NMDA
neurotoxicities. Furthermore, a better dosage ratio of TP and M (TPM10,
TP 30 mg/kg/day, M 3 mg/kg/day dose ratio is 10:1) have been designed.
TPM10 by itself did not affect the normal neurobehavioral activities but
it could exhibit the best potential efficacies against NMDA
excitotoxicities after long term administration, suggesting that TPM10
possessed a beneficial property for management of neurodegenerative
disease such as Alzheimer's diseases. Because both M and TP are currently
clinical useful drugs, this novel regimen TPM10 would be safe and
exhibited a promising novel regimen in increasing therapeutic efficacies
and attenuating adverse effects of M for clinical patients.

Patent applications by Jen-Kun Lin, Taipei TW

Patent applications by Shoei-Yn Lin-Shiau, Taipei TW

Patent applications by NATIONAL TAIWAN UNIVERSITY

Patent applications in class Bicyclo ring system having the hetero ring as one of the cyclos (e.g., chromones, etc.)

Patent applications in all subclasses Bicyclo ring system having the hetero ring as one of the cyclos (e.g., chromones, etc.)